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Site-Specific Immobilization of the Peptidoglycan Synthase PBP1B on a Surface Plasmon Resonance Chip Surface.
Van't Veer, Inge L; Leloup, Nadia O L; Egan, Alexander J F; Janssen, Bert J C; Martin, Nathaniel I; Vollmer, Waldemar; Breukink, Eefjan.
Afiliação
  • Van't Veer IL; Department of Membrane Biochemistry and Biophysics, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands.
  • Leloup NO; Crystal and Structural Chemistry, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands.
  • Egan AJ; The Centre for Bacterial Cell Biology, Newcastle University, Richardson Road, NE2 4AX, Newcastle upon Tyne, UK.
  • Janssen BJ; Crystal and Structural Chemistry, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands.
  • Martin NI; Department of Chemical Biology and Drug Discovery, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Universiteitsweg 99, 3584 CG, Utrecht, The Netherlands.
  • Vollmer W; The Centre for Bacterial Cell Biology, Newcastle University, Richardson Road, NE2 4AX, Newcastle upon Tyne, UK.
  • Breukink E; Department of Membrane Biochemistry and Biophysics, Utrecht University, Padualaan 8, 3584 CH, Utrecht, The Netherlands.
Chembiochem ; 17(23): 2250-2256, 2016 12 02.
Article em En | MEDLINE | ID: mdl-27709766
Surface plasmon resonance (SPR) is one of the most powerful label-free methods to determine the kinetic parameters of molecular interactions in real time and in a highly sensitive way. Penicillin-binding proteins (PBPs) are peptidoglycan synthesis enzymes present in most bacteria. Established protocols to analyze interactions of PBPs by SPR involve immobilization to an ampicillin-coated chip surface (a ß-lactam antibiotic mimicking its substrate), thereby forming a covalent complex with the PBPs transpeptidase (TP) active site. However, PBP interactions measured with a substrate-bound TP domain potentially affect interactions near the TPase active site. Furthermore, in vivo PBPs are anchored in the inner membrane by an N-terminal transmembrane helix, and hence immobilization at the C-terminal TPase domain gives an orientation contrary to the in vivo situation. We designed a new procedure: immobilization of PBP by copper-free click chemistry at an azide incorporated in the N terminus. In a proof-of-principle study, we immobilized Escherichia coli PBP1B on an SPR chip surface and used this for the analysis of the well-characterized interaction of PBP1B with LpoB. The site-specific incorporation of the azide affords control over protein orientation, thereby resulting in a homogeneous immobilization on the chip surface. This method can be used to study topology-dependent interactions of any (membrane) protein.
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Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Ressonância de Plasmônio de Superfície / Proteínas de Escherichia coli / D-Ala-D-Ala Carboxipeptidase Tipo Serina / Proteínas de Ligação às Penicilinas / Peptidoglicano Glicosiltransferase / Proteínas Imobilizadas Idioma: En Revista: Chembiochem Assunto da revista: BIOQUIMICA Ano de publicação: 2016 Tipo de documento: Article País de afiliação: Holanda

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Ressonância de Plasmônio de Superfície / Proteínas de Escherichia coli / D-Ala-D-Ala Carboxipeptidase Tipo Serina / Proteínas de Ligação às Penicilinas / Peptidoglicano Glicosiltransferase / Proteínas Imobilizadas Idioma: En Revista: Chembiochem Assunto da revista: BIOQUIMICA Ano de publicação: 2016 Tipo de documento: Article País de afiliação: Holanda